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Selective Blockade of Herpesvirus Entry
Mediator−B and T Lymphocyte Attenuator
Pathway Ameliorates Acute
Graft-versus-Host Reaction
Maria-Luisa del Rio, Nick D. Jones, Leo Buhler, Paula
Norris, Yasushi Shintani, Carl F. Ware and Jose-Ignacio
Rodriguez-Barbosa
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2012 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2012; 188:4885-4896; Prepublished online 6
April 2012;
doi: 10.4049/jimmunol.1103698
http://www.jimmunol.org/content/188/10/4885
The Journal of Immunology
Selective Blockade of Herpesvirus Entry Mediator–B and
T Lymphocyte Attenuator Pathway Ameliorates Acute
Graft-versus-Host Reaction
Maria-Luisa del Rio,* Nick D. Jones,† Leo Buhler,‡ Paula Norris,x Yasushi Shintani,{
Carl F. Ware,x and Jose-Ignacio Rodriguez-Barbosa*
A
ttenuation of the immune response is an obligated clinical
intervention in the treatment of acute graft rejection and
for the maintenance of long-term allograft survival as
well as for the treatment of acute relapsing episodes of autoimmunity
and in chronic autoimmune diseases (1, 2). Temporal and coordinate expression of membrane-bound receptors and soluble factors
modulates the course of the immune response during an inflammatory process. Costimulation blockade of receptor/ligand interactions that participate in the exchange of information between
APCs and T cells leads to the attenuation of the immune response
due to the impaired communication between these two cell types.
This approach represents a rational and promising therapeutic intervention to mitigate the deleterious consequences of the immune
response in transplanted patients, including those undergoing graft-
*Immunobiology Section, Institute of Biomedicine, University of Leon, 24007 Leon,
Spain; †Transplantation Research Immunology Group, Nuffield Department of Surgical Sciences, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU,
United Kingdom; ‡Surgical Research Unit, Department of Surgery, University Hospital Geneva, 1211 Geneva 14, Switzerland; xInfectious and Inflammatory Diseases
Center, Laboratory of Molecular Immunology, Sanford|Burnham Medical Research
Institute, La Jolla, CA 92037; and {Takeda Pharmaceutical, Muraoka-Higashi, Fujisawa, Kanagawa, 251-8555, Japan
Received for publication December 19, 2011. Accepted for publication March 11,
2012.
This work has been supported by Grants FIS PI#10/01039 (Fondo de Investigaciones
Sanitarias, Ministry of Health, Spanish Government) and LE007A10-2 (Department
of Education of the Regional Government, Junta de Castilla y Leon) to J.-I.R.-B.
Address correspondence and reprint requests to Prof. Jose-Ignacio RodriguezBarbosa, Immunobiology Section, Institute of Biomedicine, University of Leon,
24007 Leon, Spain. E-mail address: [email protected]
Abbreviations used in this article: BM-DC, bone marrow-derived dendritic cell;
BTLA, B and T lymphocyte attenuator; CHO, Chinese hamster ovary; CRD,
cysteine-rich domain; GvHD, graft-versus-host disease; GvHR, graft-versus-host reaction; HVEM, herpesvirus entry mediator; Ig SF, Ig superfamily; KO, knockout;
LIGHT, lymphotoxin that exhibits inducible expression and competes with HSV
glycoprotein D for HVEM, a receptor expressed by T lymphocytes; SA, streptavidin;
sBTLA.Ig, soluble mouse B and T lymphocyte attenuator bound to mouse IgG2a Fc
fragment; shLIGHT, soluble human LIGHT; smLIGHT, soluble murine LIGHT.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103698
versus-host disease (GvHD) after bone marrow transplantation, and
those suffering from autoimmune diseases (3).
Two major families of molecules are involved in the control of
T cell activation, differentiation, and survival of terminally differentiated T cells, Ig superfamily (Ig SF) and the TNF/TNFR
superfamily (4–7). In the early phase of T cell activation, interactions between molecules of the Ig SF predominate, whereas in
the late phase of T cell activation, interactions between members
of the TNF/TNFR superfamily molecules become responsible for
the maintenance of the T cell response (5, 8). Herpesvirus entry
mediator (HVEM; TNFRSF14) is widely expressed on hematopoietic and nonhematopoietic cells (9, 10), whereas B and T
lymphocyte attenuator (BTLA) expression is more restricted to the
hematopoietic cellular compartment (11–14). HVEM is a type I
transmembrane molecule containing an extracellular domain
composed of four cysteine-rich domains (CRD) (9, 15, 16) with
distinct binding sites for its ligands. BTLA and CD160 bind to
CRD1 domain of HVEM and compete with HSV gD for binding
to this receptor (17, 18), whereas the binding site for lymphotoxin
that exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes
(LIGHT; TNFSF14) is located at CRD2 and CRD3 domains of
HVEM. Topographically, BTLA/CD160 and LIGHT interact with
HVEM on opposite faces of its extracellular domain (19). CRD1 is
an essential domain for the inhibitory function of soluble HVEMIg, because its deletion results in costimulation instead (17).
HVEM represents a molecular switch depending on whether
HVEM is functioning as a ligand of BTLA/CD160 (coinhibition)
or LIGHT (costimulation) or a receptor of these molecules during
the course of an immune response (costimulation). BTLA and
CD160 engagement by HVEM expressed on the same cell (cis
interaction) transmits inhibitory signals to resting lymphocytes
and provides an intrinsic regulatory mechanism for T cell inhibition by impeding HVEM from receiving signals from the surrounding microenvironment (17, 18, 20), whereas engagement of
HVEM by LIGHT in trans delivers T cell costimulatory signals
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The cosignaling network mediated by the herpesvirus entry mediator (HVEM; TNFRSF14) functions as a dual directional system
that involves proinflammatory ligand, lymphotoxin that exhibits inducible expression and competes with HSV glycoprotein D for
HVEM, a receptor expressed by T lymphocytes (LIGHT; TNFSF14), and the inhibitory Ig family member B and T lymphocyte
attenuator (BTLA). To dissect the differential contributions of HVEM/BTLA and HVEM/LIGHT interactions, topographicallyspecific, competitive, and nonblocking anti-HVEM Abs that inhibit BTLA binding, but not LIGHT, were developed. We demonstrate that a BTLA-specific competitor attenuated the course of acute graft-versus-host reaction in a murine F1 transfer semiallogeneic model. Selective HVEM/BTLA blockade did not inhibit donor T cell infiltration into graft-versus-host reaction target
organs, but decreased the functional activity of the alloreactive T cells. These results highlight the critical role of HVEM/BTLA
pathway in the control of the allogeneic immune response and identify a new therapeutic target for transplantation and autoimmune diseases. The Journal of Immunology, 2012, 188: 4885–4896.
4886
Materials and Methods
Mice and rats
Twelve- to 16-wk-old female Lewis rats (Harland) and 8- to 12-wk-old
female C57BL/6 (B6), BALB/c (Charles River), and CB6F1 mice (offspring of BALB/c 3 B6, H-2d/b) were bred at the animal facility of the
University of Leon. All experiments with rodents were handled and cared
for in accordance with the Ethical Committee for Animal Research of the
School of Veterinary Medicine (University of Leon) and the European
Guidelines for Animal Care and Use of Laboratory Animals.
Cloning and expression of membrane-bound murine HVEM
and their soluble ligands
Total RNA was extracted from B6 splenocytes using the RNeasy mini kit
(Qiagen). Reverse transcription from mRNA to cDNA was performed with
GeneAmp RNA PCR kit (Applied Biosystems). The cloning of soluble
mouse B and T lymphocyte attenuator bound to mouse IgG2a Fc fragment
(sBTLA.Ig) recombinant protein has been previously reported (24).
Full-length murine HVEM gene was PCR amplified with a proofreading
Taq PFU polymerase and cloned into a modified pcDNA3.1+ vector
(Invitrogen), upstream of the gene encoding for monster GFP (Clontech)
inserted with EcoRV and XbaI restriction flanking sites. Flag-tagged soluble human LIGHT (hereafter, Flag-shLIGHT) and Flag-Foldon–tagged
soluble murine LIGHT (from now on, Flag-Foldon-smLIGHT) (provided
by C.F. Ware, La Jolla, CA, and Y. Shintani, Osaka, Japan, respectively)
were used for the binding experiments (25).
Cell transfection
Chinese hamster ovary (CHO) cells were seeded on 6-well plates at 2.5 3
105 cells/well in complete RPMI 1640 medium containing 10% FCS,
2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 50 mg/ml
gentamicin, and 5 3 1025 M 2-ME, and allowed to grow until they
reached 60–70% confluence. The pSecTag2 Hygro b vector (Invitrogen)
containing the extracellular domain of murine BTLA, Flag-shLIGHT and
Flag-Foldon-smLIGHT, and HVEM-monster GFP constructs was purified
using endotoxin-free Maxi-prep kit (Qiagen) and then transfected into
CHO cells with 2 mg DNA/well of each construct/liposome complex
(lipofectamine; Invitrogen) for 6–16 h (26).
Generation, characterization, and purification of anti-murine
HVEM mAbs for in vivo use
Female Lewis rats were immunized i.p. with 0.5 ml of a 1:1.2 mixture of 5–
10 3 106 HVEM stably transfected CHO cells expressing the membranebound murine HVEM-GFP fusion protein in IFA (Sigma-Aldrich). Six
weeks after the first immunization, the animals were inoculated i.v. with
10 3 106 HVEM-transfected CHO cells and the hybridoma fusion protocol
was carried out 3 d later, as previously described (24, 27). Twelve days after
the fusion, culture supernatants from growing hybridomas were collected
from 96-well plates and tested by flow cytometry against murine HVEMGFP–transfected and control GFP-transfected CHO cells for 2 h at 37˚C,
washed, and subsequently incubated with an optimal dilution of Cy5-
labeled polyclonal mouse anti-rat IgG (H+L; Jackson ImmunoResearch
Laboratories).
Hybridomas secreting anti-HVEM mAbs and isotype control rat IgG2a
(anti-plant cytokinin, clone AFRC-MAC-157, ECACC 93090997) were
purified through a protein G-Sepharose affinity chromatography, quantified,
filtered through 0.45 mm, and stored frozen at 1 mg/ml.
Flow cytometry-based competition-binding assays
A flow cytometry competition assay was established to define the epitopes
recognized by anti-HVEM Abs. We first compared each possible pair of
anti-HVEM mAbs, in which one of the Abs, the competitor, was unlabeled,
whereas the other member of the pair, the developer, was biotinylated. Thus,
by comparing each possible pair combination of anti-HVEM mAbs, we
could determine whether the Abs competed with each other for binding to
either identical or overlapping epitopes, or alternatively were recognizing
distinct epitopes located at the extracellular domain of HVEM. Each unlabeled anti-HVEM mAb was confronted to the rest of biotinylated antiHVEM mAbs of the panel. Thus, a saturating amount (10 mg) of each
unlabeled anti-HVEM mAb (competitor Ab) or rat IgG2a isotype control
was first incubated with 2.5 3 105 HVEM-GFP–transfected CHO cells,
and each of the biotinylated anti-HVEM members of the panel was added
later (developer Ab) to the immunological reaction.
To assess to what extent anti-HVEM mAbs generated in the present
studies could interfere with the binding of murine sBTLA-mouse IgG2a.Fc
(from now on sBTLA-Ig) (24), Flag-Foldon-smLIGHT or Flag-shLIGHT
fusion proteins to HVEM-GFP–transfected CHO cells, each unlabeled
anti-HVEM mAb, were first incubated with HVEM-transfected cells at
saturating concentrations (10 mg Ab per 2.5 3 105 cells) for 30 min at
room temperature. Then, in the presence of each anti-HVEM Ab as
competitor, 10 mg of each soluble recombinant fusion protein was added
and incubated for 2 h at 37˚C, the supernatant was washed out, and the
immunostaining was developed with either a biotinylated rat anti-mouse
IgG2a isotype-specific mAb (R19-15; BD Biosciences) or biotinylated
anti-Flag (clone M2; BioLegend), followed by streptavidin (SA)-PE.
GM-CSF–mediated bone marrow-derived dendritic cell
differentiation
Syngeneic C57BL/6 (B6) and allogeneic BALB/c bone marrow cells were
harvested from tibiae and differentiated with 30 ng/ml murine GM-CSF
(Peprotech), as previously described (28). Bone marrow-derived dendritic cells (BM-DC) were finally matured upon overnight exposure to 1
mg/ml LPS O111:B4 (Sigma-Aldrich).
Parental into nonirradiated F1 acute GvHR murine model of
alloreactivity
Donor B6 splenocytes were harvested, red cells were lysed, and cell suspensions were washed and resuspended in Dulbecco’s PBS. Then, 70 3 106
cells were adoptively transferred i.v. into CB6F1 recipient mice. Control
mice were injected i.v. with 70 3 106 syngeneic F1 splenocytes. All donor
cell suspensions were injected on the same day using cells processed simultaneously under the same conditions. F1 recipient mice received a
single dose of 1 mg anti-HVEM mAb or isotype-matched rat IgG2a control
administered i.p. Nineteen days after the adoptive transfer of donor B6
splenocytes into F1 recipients, mice were euthanized and the absolute
number of hematopoietic cells was counted in each lymphoid compartment. Parental cell engraftment was assessed in distinct lymphoid compartments by flow cytometry. Single-cell suspensions were first incubated
with Fc blocker (FcgRIII, clone 2.4G2) to prevent nonspecific staining,
and then cells were stained with FITC-conjugated anti–H-2d (SF1-1.1) and
Alexa 647-conjugated anti-H-2b (AF6-88.5). To further identify the different cell subsets, the following list of lineage-restricted Abs was used:
anti-CD3 (145-2C11), anti-CD4 (L3T4), anti-CD8a (53-6.7), anti-CD19
(1D3), anti-Ly6G (1A8), and anti-Ly6C (Monts-1; provided by E. Butcher,
Stanford University School of Medicine). All of these mAbs were purchased from BioLegend, except 2.4G2 and anti-Ly6C, which were purified
and labeled in our laboratory.
In all flow cytometry experiments, dead cells were excluded by propidium iodide or DAPI staining. Flow cytometry acquisition was carried out
on a Cyan 9 cytometer (Beckman Coulter). Data analysis was performed
using the WinList 3D Version 7 (Verity Software House, Topsham, ME).
CD107a degranulation assay and frequency of
IFN-g–secreting T cells
Sixteen days after GvHR induction, 2 3 105 splenocytes were isolated from
isotype control, 1H7-, and 6C9-treated F1 mice, and were restimulated
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(21, 22). An extra level of complexity to this cosignaling pathway
was found in the ability of BTLA and CD160 to function as activating ligands of HVEM in trans-promoting T cell survival (22,
23). These authors demonstrated that engagement of HVEM by
BTLA and CD160 agonist ligands induces IkBa degradation and
activation of NF-kB RelA (p65) in epithelial and T cell subsets
promoting their survival (22). Likewise, engagement of LIGHT on
activated T cells by HVEM expressed in other T cell types costimulates their T cell proliferation (22, 23).
The dual specificity of the soluble receptors, LTbR-Fc or
HVEM-Fc, leaves the interpretation of the most significant ligandreceptor pathway ambiguous. To overcome these difficulties and
dissect better the role of each interaction separately and thus
determine to which extent each interaction pathway is contributing to disease outcome, anti-HVEM mAbs that disrupt individual
ligand-receptor interaction were developed. We demonstrate
in this study that mAbs topographically specific for HVEM/
BTLA interaction are capable of ameliorating graft-versus-host
reaction (GvHR) by mitigating donor-alloreactive T cell effector
function.
HVEM/BTLA BLOCKADE PREVENTS ACUTE GvHR
The Journal of Immunology
4887
in vitro with 1 3 105 either syngeneic (B6) or allogeneic BALB/c mature
BM-DC. PE-conjugated anti-CD107a mAb (clone 1D4B; BioLegend) was
added to the MLC and incubated for 1 h at 37˚C in 5% CO2. Cells were
incubated for an additional period of 4 h in the presence of 2 mM monensin
and analyzed following CD107a degranulation assay gating on donor
CD8+ T cells (29).
To determine the frequency of CD8+ T cells secreting IFN-g, 2 3 105
splenocytes were restimulated in vitro with 1 3 105 either syngeneic (B6)
or allogeneic BALB/c mature BM-DC in the presence of monensin. Cell
cultures were then harvested, and donor CD8+ T cells were stained with
FITC-labeled anti-CD8a and PE-labeled anti-Kd. Cells were then fixed for
20 min at room temperature (fixation buffer; BioLegend), centrifuged, and
washed twice in permeabilization buffer (BioLegend). Finally, cells were
stained with allophycocyanin-labeled anti-mouse IFN-g (clone XMG1.2;
BioLegend), washed, and collected for analysis. To confirm the specificity
of the staining, cells were preincubated with unlabeled anti-mouse IFN-g
(clone XMG1.2; BioLegend) before allophycocyanin-labeled anti-mouse
IFN-g mAb was added (30).
In vivo cytotoxicity assay
Quantification of Th1/Th2 cytokine by cytometric bead array
A total of 70 3 106 parental B6 splenocytes was injected i.v. into F1
recipients, which were treated at day 0 with a single saturating dose of 1
mg either isotype-matched control (rat IgG2a) or anti-HVEM mAbs.
Sixteen days after treatment, 2 3 105 splenocytes from each experimental
group were harvested and cocultured with 1 3 105 syngeneic B6 or allogeneic BALB/c BM-DC. Supernatants were collected and analyzed at
48 h after in vitro restimulation, and the amount of IL-2, IL-4, IL-5, IFN-g,
and TNF-a was quantified using a cytometric bead array following the
manufacturer’s instructions (BD Biosciences).
Statistical analysis
Data are expressed as mean 6 SD. Statistical significance was assessed
using the parametric Student t test and nonparametric tests. A p value
,0.05 was considered statistically significant.
Results
Specificity and in vivo nondepleting activity of anti-HVEM
mAbs recognizing the extracellular domain of murine HVEM
receptor
A panel of four anti-HVEM mAbs (clones 1H7, 5B7, 6C9, and
10F3, all of them rat IgG2a, k L chain) was obtained and characterized in the initial part of this study. The specificity of antiHVEM mAbs was demonstrated via flow cytometry by their capacity to recognize CHO-HVEM-GFP–transfected cells, but not
GFP-transfected CHO cells used as negative control (Fig. 1A).
To gain further insight into the potential in vivo functional
activity of the anti-HVEM mAbs and to rule out any possible
depleting activity, anti-HVEM Abs used in the in vivo studies were
injected i.p. into F1 mice. This in vivo assay allowed us to determine simultaneously any depleting activity mediated by either
complement- or Ab-dependent cellular cytotoxicity on T or
B lymphocytes. No significant detectable decay in lymphocyte
FIGURE 1. Anti-HVEM mAbs specifically recognize HVEM receptor on transfected cells. (A) The complete HVEM-encoding gene fused
in frame to monster GFP was cloned into the mammalian pcDNA3.1
expression vector. pcDNA3.1-HVEM-GFP plasmid and empty vector
pcDNA3.1-GFP were transiently transfected into CHO cell line and
stained with rat anti-mouse HVEM mAbs, followed by Cy5-labeled mouse
anti-rat IgG polyclonal conjugate. Cells were gated on propidium iodide2
GFP+ and analyzed by flow cytometry. Dotted lines indicate control CHOGFP cell line incubated with each anti-HVEM mAb. Solid lines show the
reactivity pattern of anti-HVEM hybridoma supernatants (1H7 and 5B7,
upper panel; 6C9 and 10F3, lower panel) against HVEM-transfected CHO
cells. (B) In vivo treatment with anti-HVEM mAbs (clones 1H7 and 6C9)
that were selected for the in vivo experiments depleted neither T cells nor
B cells. F1 mice were i.p. injected with a single dose of 1 mg rat IgG2a
isotype control (black squares), 1H7 mAb (black triangles), or 6C9 mAb
(black diamonds) anti-HVEM mAbs. Total cell number of CD4, CD8, and
CD19 cells was analyzed 5 d later in spleen.
numbers 5 d after the i.p. injection of 1 mg anti-HVEM mAbs
was observed, which was in agreement with the lack of depleting
activity reported for the majority of rat Abs of this particular
isotype (33) (Fig. 1B).
Anti-HVEM mAbs within group II exhibit competitive
inhibition of sBTLA-Ig binding to HVEM-transfected cells,
without preventing HVEM/LIGHT interaction
The extracellular domain of HVEM exhibits two opposite regions
in its spatial conformation for the interaction with its ligands
BTLA/CD160 or LIGHT (19, 34). A competitive binding assay
was designed to dissect whether the panel of anti-HVEM mAb
raised in our study was recognizing the same, overlapping, or
different epitopes on the extracellular domain of HVEM. As
shown in Fig. 2, anti-HVEM mAbs were classified into two distinct epitopes based on their competitive behavior, as follows:
group I included clones 1H7 and 5B7 (Fig. 2A) and group II, 6C9,
or 10F3 (Fig. 2B).
This panel of anti-HVEM mAbs showed differential competition
with HVEM ligands. sBTLA-Fc construct specifically bound to
HVEM-transfected cells, but not to control GFP-transfected cells
(Fig. 3A) (17–19, 24, 35). Anti-HVEM Abs within group I (clones
1H7 and 5B7) did not compete with sBTLA-Fc (nonblocking Abs)
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Spleens from B6, BALB/c, and F1 mice were collected, and single-cell
suspensions were prepared in RPMI 1640 complete medium. Target cells
were washed twice in Dulbecco’s PBS and labeled with CFSE (Molecular
Probes) at 10 mM (F1), 2 mM (BALB/c), or 0.4 mM (B6) for 10 min at
37˚C. The reaction was stopped by adding 2 vol of cold RPMI 1640
containing 10% FCS, followed by two washes in Dulbecco’s PBS. A total
of 30 3 106 of each target cell (B6, BALB/c, and F1 cells) was mixed at
ratio 1:1:1 and was subsequently i.v. injected into F1 recipients at day 16
post-GvHR induction. Three days later (day 19 post-GvHR induction),
recipient F1 mice were euthanized and target cells were analyzed in spleen
and peripheral lymph nodes. The percentage of specific target lysis was
calculated by comparing the survival of each target population with the
survival of syngeneic population according to the following equation:
percentage of specific killing of target cells = 100 2 ([absolute number of
target population in experiment/absolute number of syngeneic population
in experiment]/[absolute number of target population in F1 recipient/
absolute number of syngeneic population in F1 recipient]) 3 100 (31, 32).
4888
(Fig. 3B, upper panel), although group II, clones 6C9 and 10F3,
completely abrogated sBTLA-Ig binding to HVEM on transfected
cells (blocking Abs) (Fig. 3B, lower panel).
Flag-soluble human LIGHT recombinant fusion proteins bound
specifically to HVEM-transfected cells in flow cytometry (Fig. 3C).
Neither group I nor II anti-HVEM Abs (Fig. 3D) interfered with
Flag-shLIGHT binding to HVEM. Similar results were obtained
with mouse version Flag-Foldon soluble LIGHT recombinant fusion proteins (Fig. 3E, 3F).
Altogether, the data indicate that the set of anti-HVEM Abs
mapped to two topographically different epitopes on the extracellular domain of HVEM. The anti-HVEM mAbs in group II
that blocked HVEM-BTLA interaction may be the most suitable therapeutic tool for the in vivo evaluation of the biological
consequences derived from selective Ab-mediated blockade of
HVEM/BTLA interaction.
Donor antihost alloreactive T cells downmodulate HVEM
receptor expression following alloantigen recognition
To monitor the impact of the alloreactive T cell response on the
modulation of HVEM receptor expression, B6 splenocytes were
adoptively transferred to F1 recipients (B6-F1), whereas baseline
expression of HVEM was monitored in syngeneic F1 recipients
adoptively transferred with F1 splenocytes (F1-F1). Under homeostatic conditions, naive resting B cells expressed HVEM to
a lower extent than T cells (Fig. 4A), which is in agreement with
previous reports (14, 36, 37). The expression of HVEM was
quickly downregulated on alloreactive CD4 and CD8 T cells as
soon as day 3 after the adoptive transfer, and the reduced expression was sustained when compared with the amount of HVEM
expressed on host T cells of syngeneic recipients at the same time
points (Fig. 4A, 4B). Interestingly, expression of HVEM on host
T cells of F1 recipients adoptively transferred with allogeneic B6
splenocytes did not undergo any apparent change in HVEM expression (Fig. 4A, 4B). Indeed, the amount of HVEM expressed
on host T cells of F1 recipients was similar regardless of whether
they were adoptively transferred with either syngeneic F1 or allogeneic B6 splenocytes. In contrast, BTLA was reciprocally upregulated early after T cell activation, and its expression then
declined gradually and more rapidly on CD8 T cells than on CD4
T cells (Fig. 4C).
These observations indicate that bystander stimulation by the
inflammatory milieu created by donor alloreactive T cells reacting
against host tissues did not influence HVEM expression on host
T cells or B cells. More importantly, modulation of HVEM and
BTLA following donor T cell activation was dependent on TCR
recognition of host alloantigens. These data support the idea that
HVEM/BTLA cis prevents nonspecific activation in an inflammatory milieu.
Blockade of HVEM/BTLA ameliorates the course of rejection
of host hematopoietic cells during the acute phase of the GvHR
The adoptive transfer of donor B6 splenocytes into nonirradiated
F1 recipients (B6 3 BALB/c) induces a progressive alloreactive
response against host BALB/c H-2d histoincompatible Ags (38).
Flow cytometry was used to measure the loss of host hematopoietic cells in primary hematopoietic (bone marrow and thymus)
and secondary (spleen) organs using a combination of Alexa 647labeled anti-murine Kb and FITC-labeled anti-murine Kd.
We examined the panel of mAbs to mouse HVEM to address the
question of whether epitope-selective HVEM blockade will attenuate the GvHR. An increase in host H-2d–positive leukocytes
indicated that treatment of mice with blocking anti-HVEM mAb
(6C9) efficiently protected the host bone marrow compartment
from donor T cell-mediated rejection in the acute phase of GvHR.
In contrast, a dramatic loss of H-2d cells in the bone marrow was
observed in F1 mice receiving semiallogeneic splenocytes treated
with either nonblocking anti-HVEM mAb (clone 1H7) or rat
IgG2a isotype control (Fig. 5A). This result was a clear indication
that competitive blockade of HVEM/BTLA interaction was sufficient to attenuate the course of graft rejection in host bone
marrow cells. The global prevention of host bone marrow rejection after 6C9 Ab-mediated blockade was also reflected in different host leukocyte subsets present in this compartment, when
compared with isotype- or 1H7-treated F1 recipient mice receiving
B6 donor allogeneic splenocytes. Thus, host granulocytes (Fig.
5B) and monocytes (Fig. 5C) were significantly more protected in
6C9-treated F1 mice than in 1H7- or isotype-treated allogeneic F1
recipients. Treatment with blocking anti-HVEM mAb (clone 6C9)
conferred substantial, but incomplete protection against rejection
of B cells when compared with B cells in the syngeneic F1-F1
recipients (Fig. 5D).
Host thymocytes and double-positive thymocytes were fully
protected from rejection by the blocking anti-HVEM mAb (6C9)
(Fig. 5E, 5F), as were lymphocytes in the spleen (Fig. 5G).
These data indicate that blockade of HVEM/BTLA interaction
mitigates the acute phase of GvHR by attenuating the rejection of
host hematopoietic cells.
Blockade of HVEM/BTLA interaction reduces the frequency of
donor CD8+ T cells expressing CD107a and secreting IFN-g
To dissect the contribution of HVEM/BTLA interaction to the
course of GvH development, donor T cell infiltration on distinct
hematopoietic target tissues was assessed. We found that CD4+ and
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FIGURE 2. The panel of anti-HVEM mAbs defines the existence of at
least two distinct epitopes on the extracellular domain of HVEM. With the
purpose of mapping epitopes on HVEM receptor, HVEM-transfected cells
(2 3 105 cells/well) were incubated with a saturating amount (10 mg/well)
of each unlabeled anti-HVEM mAb or isotype-matched control. AntiHVEM mAbs were classified within group I (clones 1H7 and 5B7) (A) or
group II (clones 6C9 and 10F3) (B) based on their competition profile.
Stably transfected HVEM-GFP CHO cells were incubated for 1 h at 4˚C
with a saturating amount of 10 mg/well of each unlabeled anti-murine
HVEM mAb (competitor Ab). To detect competition among Abs recognizing the same molecule, each biotinylated anti-HVEM Ab (developer
Ab, solid lines) was added to HVEM-transfected CHO cells, and the immunological reactions were revealed using an optimal dilution of SA-PE.
Dashed lines depict HVEM-transfected cells incubated with an irrelevant
competitor isotype-matched rat IgG2a control. The two first rows of dot
plots display no competition profiles, whereas the third row exhibits those
anti-HVEM Abs that competed with each other. One representative experiment of three with identical results is shown.
HVEM/BTLA BLOCKADE PREVENTS ACUTE GvHR
The Journal of Immunology
4889
FIGURE 3. Group II anti-HVEM mAbs exhibit antagonist activity and
abrogate HVEM/BTLA interaction without affecting HVEM/LIGHT
binding. (A) The specificity and binding affinity of recombinant sBTLA-Ig
fusion protein to membrane-bound HVEM-GFP stably transfected CHO
cells or control CHO-GFP cells are shown on gated GFP-positive cells.
Dashed line corresponds to binding of sBTLA-Ig to CHO-GFP controltransfected cells, and solid line depicts the binding of sBTLA-Ig to
HVEM-GFP–transfected cells. (B) A total of 2.5 3 105 stably HVEMGFP–transfected CHO cells was incubated with 10 mg/ml either rat IgG2a
competitor control (dashed lines) or anti-HVEM mAbs (solid lines) following group I (upper panel) or group II (lower panel) for 30 min at room
temperature. Then, 10 mg/ml sBTLA-Ig was added to the cells and incubated at 37˚C for 2 h. The reaction was washed and further incubated with
biotinylated rat anti-mouse IgG2a mAb, and the staining was finally developed with SA-PE. (C) The specificity of Flag-shLIGHT binding to
HVEM-GFP–transfected cells (solid line) is shown. Dashed line displays
the background binding of Flag-shLIGHT to control GFP-transfected cells.
(D) Stably transfected HVEM-GFP cells were incubated with 10 mg/ml
either rat IgG2a competitor Ab control (dashed lines) or anti-HVEM mAbs
(solid lines) following group I (D, upper panel) or group II (D, lower
panel) for 30 min at room temperature. Then, 10 mg/ml Flag-shLIGHT was
added to the reaction and incubated at 37˚C for 2 h. Biotinylated anti-Flag
mAb followed by SA-PE was used to develop the immunological reactions. (E) The specificity of Flag-Foldon-smLIGHT binding to HVEMGFP–transfected cells (solid line) is depicted. Dotted line represents the
background binding of Flag-Foldon-smLIGHT to control GFP-transfected
cells. (F) Stably transfected HVEM-GFP cells were incubated with 10 mg/
ml either rat IgG2a competitor Ab control (dashed lines) or anti-HVEM
mAbs (solid lines) following group I (F, upper panel) or group II (F, lower
panel) for 30 min at room temperature. Then, 10 mg/ml Flag-Foldon
smLIGHT was added to the reaction and incubated at 37˚C for 2 h. Biotinylated anti-Flag mAb followed by SA-PE was used to develop the immunological reactions. A cartoon of the competition assay setup is shown.
One representative experiment of three with identical results is shown.
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CD8+ T cell infiltration in 6C9-treated F1 recipient mice was
similar to that of isotype- or 1H7-treated F1 mice in distinct host
hematopoietic compartments. Thus, in the bone marrow, thymus,
and spleen, the absolute number of donor CD4+ or CD8+ T cells
infiltrating this tissue was similar for all experimental groups (Fig.
6). Thus, the blockade of HVEM/BTLA interaction by antiHVEM 6C9 did not impact in the absolute number of T cells
infiltrating host hematopoietic GvHR major target organs, suggesting perhaps that the effector function was blocked. To test this
idea, we measured CD107a degranulation in CD8 T cells and
expression of intracellular IFN-g as well as soluble cytokines
characteristic of differentiated Th1 and Th2 cells. Splenocytes
from anti-HVEM– or rat IgG2a-treated F1 recipients (at day 16
posttransplantation) were restimulated in vitro with allogeneic
BALB/c mature BM-DC to monitor donor antihost CD8+ T cellmediated allosensitization in response to alloantigen following the
CD107a degranulation assay. Donor-alloreactive and host CD8+
T cells were gated separately, and CD107a expression was analyzed in isotype control- and in anti-HVEM–treated F1 mice.
The frequency of CD8+ T cells expressing CD107a was suppressed in donor cells from mice treated with anti-HVEM 6C9
compared with mice receiving rat IgG2a control or the antiHVEM 1H7 mAb, which were significantly elevated compared
with the syngeneic F1/F1 control graft (Fig. 7A). Similarly, the
frequency and absolute number of IFN-g–secreting cells decreased in anti-HVEM 6C9-treated mice when compared with
controls (Fig. 7B).
IL-2, IL-4, IL-5, IFN-g, and TNF-a secreted by CD4+ Th1 and
Th2 were also measured in supernatants of splenocytes of F1
recipients adoptively transferred with B6 splenocytes, which
were restimulated in vitro with syngeneic and allogeneic mature
BM-DC for 48 h. Cytokines of Th1 cells, particularly IFN-g,
were significantly augmented in F1 mice treated with rat IgG2a
isotype or 1H7 mAbs compared with recipients treated with 6C9
mAb, but no significant differences were seen when IL-2, IL-4,
IL-5, or TNF-a was analyzed in the same in vitro restimulation
assay (Fig. 7C).
4890
HVEM/BTLA BLOCKADE PREVENTS ACUTE GvHR
These data indicate that blockade of HVEM/BTLA interaction
mitigates the course of GvHR by inhibiting the frequency and
cytotoxic function of donor-alloreactive CD8 and CD4 T cells.
In vivo donor antihost CTL activity is significantly reduced
after HVEM/BTLA blockade during the acute phase of GvHR
Donor CTL-mediated allogeneic responses play a critical role in the
rejection of host hematopoietic tissues that occurs after the adoptive
transfer of allogeneic parental splenocytes into F1 recipients (38, 39).
To investigate the consequences of in vivo donor antihost CTL response after selective HVEM/BTLA blockade, F1 recipient mice
were injected with an identical number of B6, BALB/c, and F1 target cells that were differentially labeled with different amounts
of CFSE, as described in Materials and Methods. A quantitative
analysis of the percentage of killing of target BALB/c and F1 cells in
host spleen and peripheral lymph nodes was evaluated. A significant
reduction of killing of F1 and BALB/c target cells was observed in F1
mice after selective blockade of HVEM/BTLA with 6C9 mAb
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FIGURE 4. Reciprocal regulation of HVEM and BTLA expression is dependent on alloantigen recognition by donor T cells during the course of GvHR.
(A) A total of 70 3 106 of B6 splenocytes was adoptively transferred into F1 recipients (B6-F1), and the time course of HVEM expression was monitored at
days 3, 7, 11, and 14 after GvHR induction on host and donor CD4+ T cells, CD8+ T cells, and CD19+ cells of spleen. Syngeneic adoptive transfer of F1
splenocytes to F1 recipients (F1-F1) served as control group to determine the basal level of expression of these molecules at resting state under noninflammatory conditions. Black solid lines represent basal expression of HVEM on host cells of F1 mice receiving syngeneic F1 splenocytes; black dashed
lines display isotype-matched control. Allogeneic adoptive transfer of B6 splenocytes to F1 recipients allowed the monitoring of surface expression of
HVEM receptor on host (red solid lines) and donor (blue solid lines) lymphocytes during the course of GvHR. Black dashed lines indicate isotype-matched
control. Biotinylated anti-HVEM mAb (10F3) followed by SA-PE was used to develop the reaction. The mean fluorescence intensity (MFI) of HVEM (B)
and BTLA (C) expression on CD4 and CD8 T cells was calculated at different time points after the adoptive transfer of allogeneic splenocytes to F1
recipient mice. Red open circles and blue closed circles depict values of MFI of host and donor T cells, respectively. At each time point, MFI of HVEM and
BTLA expression on donor and host CD4 and CD8 T cells was compared, and the statistical significant differences are indicated inside the plots (*p , 0.05,
**p , 0.005, ***p , 0.0005; ns, nonsignificant). Blue dotted line highlights the expression trend of HVEM and BTLA on donor CD4 and CD8 T cells.
The Journal of Immunology
4891
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FIGURE 5. Blockade of HVEM/BTLA interaction attenuates the rejection of host target tissues. A total of 70 3 106 of allogeneic donor B6 splenocytes
was adoptively transferred into F1 recipients, which were treated with a single dose of 1 mg either rat IgG2a isotype control (black squares), nonblocking
anti-HVEM mAb (1H7, black triangles), or blocking anti-HVEM mAb (6C9, black diamond) at day 0. A syngeneic control group, in which 70 3 106 F1
splenocytes were adoptively transferred to F1 recipients (black circles), was included in the experimental setup. The absolute number of host bone marrow
cells (A), granulocytes (B), monocytes (C), B cells (D), as well as the total number of host F1 thymocytes (E), host F1 double-positive thymocytes (F), and
host splenocytes (G), are depicted at 19 d after the adoptive transfer of syngeneic or allogeneic B6 splenocytes. Statistical significance and the p value were
calculated using unpaired Student t test and Mann-Whitney U test. The following criterion of significance was used, as follows: *p , 0.05, **p , 0.005,
***p , 0.0005. The experiment was repeated twice with similar results.
compared with either isotype- or 1H7-treated groups in spleen (Fig.
8A) and peripheral lymph nodes (Fig. 8B) (p , 0.0005).
Discussion
Hematopoietic stem cell transplantation and global and longlasting immunosuppression are associated with delayed recovery
of host immunocompetence, frequent opportunistic infections, and
GvHD. More specific pharmacological interventions are necessary
for the treatment of hematological disorders after bone marrow
transplantation and for the treatment of the GvHD-related side
effects (40). Molecules of the Ig SF and molecules of TNF/TNFR
superfamily play a nonredundant and complementary role in
T cell activation, differentiation, and the acquisition of effector
function (41, 42).
In this study, we define a panel of topographically distinct Abs to
mouse HVEM that segregate into two groups, as follows: HVEM-
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HVEM/BTLA BLOCKADE PREVENTS ACUTE GvHR
BTLA competitive group and noncompetitive group; neither group
competes with the binding of LIGHT. We demonstrate that the
competitive blocking HVEM Ab 6C9 suppresses the immune rejection in an allogeneic GvHR murine model. Our results indicate
the effector mechanisms of CD4 and CD8 T cells require HVEM/
BTLA signaling.
The importance and scientific relevance of the role of HVEM/
BTLA/CD160 and HVEM/LIGHT interaction in transplantation
and autoimmune disease have been evaluated in vitro and in vivo
using several experimental approaches. The costimulatory function of the HVEM receptor has been revealed in transplantation
experiments, in which graft rejection was attenuated, such as in
allogeneic donor infusion of HVEM or LIGHT knockout (KO)
T cells to lethally irradiated histoincompatible hosts, rescued
with a syngeneic or allogeneic bone marrow transplant (43–45)
or transplantation of MHC-mismatched tissues into HVEM or
LIGHT KO recipients (46). In line with the costimulatory function
of HVEM, Xu et al. (43) have provided evidence that Abmediated blockade of both HVEM/BTLA and HVEM/LIGHT
interactions with an antagonist hamster anti-HVEM mAb (clone
LBH1) in lethally irradiated mice that were rescued with allogeneic T cell-depleted bone marrow cells plus allogeneic splenocytes effectively protected host hematopoiesis from rejection in
various bone marrow transplantation settings across distinct histocompatibility barriers. However, these results did not definitely
elucidate the dilemma of whether HVEM/BTLA or HVEM/
LIGHT blockade was the most crucial interaction for the prevention of disease and to which extent each pathway acting separately contributes to the overall protective effect of GvHD (43).
In their studies, LBH1 Ab-mediated blockade of both HVEM/
BTLA and HVEM/LIGHT pathways would prevent all possible
signals through HVEM receptor (19, 22). In a parallel setting,
these authors also adoptively transferred allogeneic HVEM KO or
LIGHT KO splenocytes to semiallogeneic nonirradiated or irradiated F1 recipients (43), but HVEM deficiency precludes LIGHT
and BTLA costimulatory signaling in trans through HVEM, and
also prevents HVEM from delivering negative signals upon engaging BTLA. The same applies to donor LIGHT KO T cells that
cannot receive signals from HVEM or LTbR or costimulate other
T cells through HVEM (43, 47–50).
The use of decoy receptors and receptor-specific–deficient
mice to address the role of complex pathways of interactions, in
which multiple ligand/receptor cross-interactions are involved, provides limited mechanistic information. An interpretative dilemma
often emerges, for instance, with the use of soluble HVEM-Ig that
would interfere with both HVEM/BTLA/CD160 coinhibitory/
costimulatory interactions and also HVEM/LIGHT costimulatory
axis. The same occurs with the use of donor HVEM KO or LIGHT
KO T cells and HVEM KO or LIGHT KO mice as recipients,
because all potential bidirectional interactions of HVEM expressed
on hematopoietic and nonhematopoietic cells with their ligands are
completely abolished. Therefore, the in vivo consequences derived
from these experimental settings should be taken with caution
before drawing definitive conclusions.
To overcome these difficulties and gain insight into this complex network of HVEM interactions, we propose the utilization of
highly specific nondepleting mAbs against epitopes located at the
extracellular domain of HVEM permitting the targeting of only one
potential interaction, without perturbing other possible interactions
between HVEM and its ligands. Following these premises, and
taking into account that BTLA and LIGHT bind to nonoverlapping
and opposite sites on HVEM molecule (LIGHT binds to CRD2/3
domains of HVEM, whereas BTLA binds to CRD1 domain of
HVEM) (18, 19), it was postulated that the specific blockade of
BTLA/HVEM could be accomplished specifically with an Abbased strategy, without perturbing HVEM/LIGHT interaction.
With that goal in mind, we have characterized a set of anti-HVEM
Abs that followed two different patterns of HVEM recognition.
Thus, anti-HVEM Abs within group II are likely recognizing an
epitope located on the binding site of interaction between domain
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FIGURE 6. Ab targeting of BTLA does not reduce donor T cell number infiltrating host hematopoietic tissues during the acute phase of GvHR.
Donor B6 splenocytes were adoptively transferred
into allogeneic F1 recipient mice treated with irrelevant isotype rat IgG2a control (black square),
nonblocking anti-HVEM mAb (clone 1H7, black
triangle), or blocking anti-HVEM mAb (clone
6C9, black diamond). The absolute number of donor CD4+ cells and CD8+ cells infiltrating host
bone marrow (A), thymus (B), and spleen (C) at
day 19 of the acute phase of GvHR is depicted. No
statistically significant differences were found between isotype- and anti-HVEM–treated F1 recipients. The experiment was repeated twice with
a similar number of mice, and similar findings
were recorded.
The Journal of Immunology
4893
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FIGURE 7. Reduced frequency and absolute number of donor alloantigen-specific CD8+ T cells expressing CD107a and secreting IFN-g, and diminished Th1 cytokine production after blockade of HVEM/BTLA interaction. (A) Sixteen days after GvHR induction, 2 3 105 splenocytes isolated from
either isotype control or anti-HVEM–treated F1 mice were restimulated in vitro with 1 3 105 allogeneic BALB/c mature BM-DC per well for 1 h in the
presence of PE-conjugated anti-CD107a mAb or PE-conjugated isotype control and additional 4 h in the presence of monensin. A five-color flow
cytometry panel was used to simultaneously analyze surface markers. Cells were stained with Alexa 647-labeled anti-mouse Kb and FITC-labeled antimouse Kd to distinguish donor and host T cells, and the percentage of each population was calculated. After gating on donor and host CD8+ T cells, the
percentage and absolute number of CD8+ T cells expressing CD107a were assessed in isotype- and anti-HVEM–treated F1 recipients. PE-labeled isotypematched nonbinding control Ig was used to set the quadrant lines. This figure is a representative experiment of two performed with similar results. (B)
Splenocytes were restimulated in vitro with allogeneic APCs, as described in (A), and then stained with FITC-labeled anti-CD8a and PE-labeled anti-Kd.
After surface staining, splenocytes were washed, fixed, and permeabilized, and intracellular IFN-g staining was performed. Quadrant lines represent
appropriate negative control to confirm the specificity of the anticytokine staining that was set preincubating splenocytes with unlabeled anti-mouse IFN-g
(blocker), followed by allophycocyanin-labeled anti-mouse IFN-g. The percentage and absolute number of donor CD8+ IFN+-g T cells in each experimental group were calculated in the absence of blocker, followed by allophycocyanin-labeled anti-mouse IFN-g. One representative experiment of two
performed is displayed in this figure. (C) A total of 2 3 105 splenocytes was collected at day 16 from isotype- or anti-HVEM–treated F1 mice undergoing
GvHR and was cocultured with 1 3 105 syngeneic B6 (white bars) or allogeneic BALB/c (black bars) BM-DC for 48 h. Cell culture supernatants were then
harvested, and Th1/Th2 cytokines were monitored by cytometric bead array. Mean fluorescence intensity values were obtained for each given cytokine,
and cytokine concentrations (pg/ml) were calculated relative to the appropriate calibration curves with standard dilutions. A significant increase of IFN-g
was found in isotype- or 1H7-treated F1 mice compared with F1 mice receiving blocking 6C9 mAb. No significant differences were seen between mice
treated with nonblocking and blocking Abs against HVEM for the rest of cytokines tested in the same assay (IL-2, IL-4, IL-5, and TNF-a). Bars indicate
mean 6 SEM, and t test was used to compare differences between groups. The degree of significance was indicated as follows: **p , 0.005, ***p ,
0.0005.
4894
CRD1 of HVEM and BTLA because they exhibited blocking
activity, whereas anti-HVEM Abs of group I would be recognizing
other epitopes than those located on the HVEM binding site of
BTLA or LIGHT. The identification of blocking and nonblocking
epitopes on the extracellular domain of HVEM provided us with
an extraordinary investigative tool to determine the functional
relevance of blocking specifically HVEM/BTLA without altering
HVEM/LIGHT binding site and determine the biological consequences in preventing GvHR development.
It should not be overlooked that HVEM/BTLA Ab blockade is
probably interrupting a bidirectional pathway of signal transduction between HVEM/BTLA. That is, both costimulatory signals
transduced in trans after the engagement of HVEM on T cells by
BTLA expressed on other immune cell types as well as coinhibitory signals transduced in cis upon interaction between HVEM
and BTLA on the same cell type might be blocked (51). Because
the outcome of HVEM/BTLA blockade is disease prevention in
our experimental setting, instead of promotion of disease development, the data support that the costimulatory function of HVEM
dominates over the coinhibitory activity. Moreover, in the context
of an allogeneic response, T cell activation upregulates BTLA
expression and downregulates HVEM expression on the same cell.
This would decompensate the stoichiometry of cis inhibitory
interactions on the same cell and would promote BTLA trans
costimulatory interactions with other surrounding cells expressing
HVEM, and Ab-mediated blockade of HVEM/BTLA would prevent them (18).
The adoptive transfer of donor allogeneic BTLA KO, HVEM
KO, or LIGHT KO splenocytes to F1 recipients has been frequently
associated with the attenuation of rejection of host hematopoietic
target tissues due to poor survival and increased apoptosis of the
donor T cells, despite donor T cell proliferation proceeding normally (43–45, 52). The use of 6C9 Ab-mediated blockade of
HVEM/BTLA in a similar murine model of GvHR led to the same
outcome, that is, protection against rejection of host hematopoietic
target tissues. This protection was not, however, accompanied by
decreased donor T cell infiltration in host hematopoietic target
tissues in 6C9-treated mice compared with those mice receiving
nonblocking anti-HVEM mAb or isotype-treated control. To reconcile the results of poor survival of HVEM-deficient donor
T cells adoptively transferred to allogeneic F1 recipients (43) and
the good survival of donor T cells after Ab-mediated blockade
of HVEM/BTLA interaction, one possible explanation is that
HVEM-deficient donor T cells would not receive costimulatory
signals from both BTLA and LIGHT, whereas 6C9 Ab treatment
would only interfere with BTLA/HVEM interaction, but would
allow the transmission of LIGHT-mediated costimulatory survival
signals upon engagement with HVEM. Moreover, blockade of
HVEM/BTLA interaction did not affect the absolute number of
T cells infiltrating the graft-versus-host target tissues, but diminished the frequency and absolute number of donor alloantigenspecific CD8 T cells secreting IFN-g or expressing CD107a
compared with those receiving nonblocking Abs or isotypematched control. Besides, the amount of IFN-g released by donor alloantigen-specific T cells was also decreased after HVEM/
BTLA blockade. These observations were in accordance with
in vivo decreased cytolytic function of donor T cells after competitive blockade of HVEM/BTLA interaction. Altogether, our
data may well account for the attenuation of the cytotoxic donor
antihost response after HVEM/BTLA blockade during the course
of parental to F1 GvHR by either affecting the differentiation
of naive CD8 T cells to effector CD8 T cells due to a decreased of
T cell help or compromising the survival and maintenance of
donor alloantigen-specific effector CD8 T cells.
Parent into nonirradiated or lethally irradiated F1 recipients have
been extensively used as preclinical models for the study of alloreactivity and bone marrow rejection. The limitations of the nonirradiated model are that splenocytes adoptively transferred to
allogeneic or semiallogeneic recipients undergo acute GvHD and
immunoincompetence is only transient, mimicking the situation in
humans under nonmyeloablative conditioning regimens (53). In
contrast, the irradiated model resembles more the myeloablative
conditioning regimens, in which allogeneic T cells adoptively
transferred along with T cell-depleted bone marrow cells lead to
development of different patterns of acute GvHD depending on
the differences in class I and class II MHC Ags between donor and
recipient, and involve the effector functional activity of CD4+ and
CD8+ T cells (54). The space created after irradiation is fully
permissive to alloreactive T cells to freely expand very quickly in
response to two type of stimuli; one is alloantigen, and the other is
homeostatic proliferation in response to an emptied hematopoietic
compartment, which induces a conversion of the phenotype of the
alloreactive and nonalloreactive T cell repertoire into memory-like
T cells with less costimulatory requirements (55, 56). Because of
those influences, T cells become more refractory to therapeutic
manipulation, as it has been demonstrated for CD28/B7 blockade
with CTLA-4.Ig (57). Another limitation of the nonirradiated
model is that skin pathology and weight loss are reduced compared with this pathology in the irradiated model. Acute GvHD in
nonirradiated model is largely manifested as immune deficiency
and mixed chimerism, with less mortality than in irradiated model
(39, 58–60). Despite the above mentioned limitations of the
nonirradiated model, this was chosen because it does not require
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FIGURE 8. Significant reduction of in vivo donor antihost cytotoxic
response after HVEM/BTLA blockade. Recipient F1 mice were adoptively
transferred with 70 3 106 of B6 splenocytes and treated with either isotype
control (rat IgG2a, 1 mg), anti-HVEM (nonblocking, clone 1H7, 1 mg), or
anti-HVEM (blocking, clone 6C9, 1 mg) mAbs. Sixteen days later, recipient mice received 30 3 106 splenocytes of each CFSE-labeled target
cell, as follows: B6 (0.4 mM), BALB/c (2 mM), and F1 (10 mM). The
percentage of specific lysis in spleen (A) and peripheral lymph node
(inguinals plus axilars) (B) was calculated at 72 h, according to the
equation described in Materials and Methods. Data are representative of
two independent experiments with three mice per group. Bars indicate
mean 6 SEM, and t test was used to compare differences between groups.
Statistical significance was indicated, as follows: ***p , 0.0005.
HVEM/BTLA BLOCKADE PREVENTS ACUTE GvHR
The Journal of Immunology
manipulation of the recipient and avoids the many variables introduced by a lymphopenic environment that affect the course
of the alloreactive T cell response. Thus, we could interrogate
the experimental system to assess the influence of HVEM/BTLA
blockade on the initiation and progression of the cytotoxic response in a peripheral environment, in which alloreactive T cells
are not homeostatically proliferating. Disease pathogenesis at day
19 after the adoptive transfer of allogeneic splenocytes is largely
dependent on alloreactive effector CD8 T cells and not on cytotoxic autoreactive Abs, which appear later along with the chronic
manifestations of the disease (38).
Therefore, our data favor the notion that attenuation of donor
antihost cytotoxicity accounts for the reduced donor antihost rejection in a parent to nonirradiated F1 murine model of GvHR
under blockade of only HVEM/BTLA interaction. This refined
Ab-based strategy presented in this work, compared with previous
studies, allowed us to demonstrate the contribution of HVEM/
BTLA pathway to the attenuation of graft rejection in a GvHR
murine model.
We are particularly grateful to Leonides Alaiz for outstanding animal husbandry. We also thank Dr. Fermı́n Sánchez-Guijo (Department of Hematology, Salamanca University Hospital, Salamanca, Spain) for suggestions
and critical reading of the manuscript.
Disclosures
The authors have no financial conflicts of interest.
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HVEM/BTLA BLOCKADE PREVENTS ACUTE GvHR